RNA editing enzyme

ABSTRACT

The present invention provides a human RNA editing enzyme (REE-2) and polynucleotides which identify and encode REE-2. The invention also provides expression vectors and host cells, agonists, antibodies, or antagonists. The invention provides methods for producing REE-2 and for treating diseases associated with expression of REE-2.

This application is a division of Ser. No. 08/816,241 filed Mar. 13,1997 now U.S. Pat. No. 5,804,185.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of anovel RNA editing enzyme and to the use of these sequences in thediagnosis, prevention, and treatment of cancer, auto immune disorders,circulatory system disorders, viral diseases, and neurological diseases.

BACKGROUND OF THE INVENTION

Apolipoprotein B (apo B) circulates in two distinct forms referred to asapo B100 and apo B48. Apo B48 is encoded by same gene as apo B100 andarises as a result of mRNA. editing. A single cytidine nucleotide in apoB100 mRNA is deaminated by a zinc-containing enzyme resulting in a CAAto UAA-stop codon change (Navaratnam N. et al. (1995) Cell 81: 187-195).RNA editing of apo B has important consequences in the catabolism ofplasma lipoproteins and the ability to generate hybrid lipoproteins(Davidson N. O. (1993) Ann. Med. 25: 539-53).

Hadjiagapiou C. et al. (1994, Nucleic Acids Res. 22: 1874-1879) clonedthe apo B mRNA editing protein (HEPR) from human small intestine cDNA.HEPR is the catalytic subunit of an enzyme complex which includes, yetto be identified, complementation factors (Teng B. et al. (1993) Science260: 1819). HEPR contains consensus phosphorylation sites as well asconserved histidine and cysteine residues identified as a Zn²⁺ bindingmotif in other cytidine deaminases. Mutational studies indicated thatthe putative zinc-coordinating residues H₆₁, C₉₃, and C₉₆ and thecatalytically active residues E₆₃ and P₉₂ are necessary for both RNAediting aid cytidine deaminase activities (MacGinnitie et al. (1995) J.Biol. Chem. 270: 14768-14775). H₆₁ is also required for RNA bindingactivity.

HEPR is expressed in the adult small intestine and to a much lesserextent in the stomach, colon, and testis (Hadjiagapiou et al, supra).The rabbit homolog of HEPR is only expressed in the small and largeintestine, and the complementation proteins, essential for RNA editing,were found to exist in a wide range of organs that do not express apo B.This suggests the presence of additional RNA editing enzymes with a morewidespread role in the generation of RNA and protein diversity (YamanakaS. et al. (1994) J. Biol. Chem. 269: 21725-21734; Hodges P. et al.(1992) Trends Biochem. Sci. 17: 77-81).

The principle of therapeutic RNA editing was demonstrated by using cellextracts containing an RNA editing enzyme to correct an aberrant stopcodon introduced into synthetic RNA encoding dystrophin protein (Woolf,T. M. et al. (1995) Proc. Natl. Acad. Sci. 92: 8298-8302). Deaminationof a nucleotide in a stop codon resulted in translation read-through anda dramatic increase in expression of a downstream gene.

Apo B editing is a mechanism which determines how much apo B48 issynthesized in place of apo B100 in a specific tissue. Apo B100 is theexclusive apolipoprotein of low density lipoproteins which transportmost of the plasma cholesterol in humans. In contrast, apo B48 isdirected to chylomicrons, triglyceride-rich lipoproteins that transportdietary lipids and undergo catabolic clearance much faster thanparticles containing apo B100 (Young, S. G. (1990) Circulation 82:1574-1594). Apo B editing has major physiological and clinicalimplications. All apo B100 containing lipoproteins are atherogenic,especially when present in high concentrations. Alterations in apo B100can cause either hypocholesterolemia or hypercholesterolemia(Innerarity, T. L. et al. (1991) Adv. Exp. Med. Biol. 285: 25-31). Apo BmRNA editing decreases the amount of apo B100 production. Somatic genetransfer of rat REPR, the rat homolog of HEPR, into the liver of miceessentially eliminates apo B100 and plasma low-density lipoproteinwithout affecting anti-atherogenic high density lipoproteiris (Teng, B.et al. (1994) J. Biol. Chem. 269: 29395-29404).

Alpha-galactosidase is a lysosomal enzyme that is deficient in patientswith Fabry's disease. After excluding other possibilities, Novo, F. J.et al. (1995, Nucleic Acids Res. 23: 2636-2640) proposed that RNAediting is responsible for an observed nucleotide conversion inalpha-galactosidase mRNA from Fabry's disease patients. The enzymeresponsible for the nucleotide conversion has not been identified.

Among the main characteristics of psoriasis are abnormal keratinocyteproliferation and differentiation. A search for proteins that areimplicated in psoriasis yielded a partial sequence for phorbolin I, aprotein upregulated in psoriatic keratinocytes (Madsen, P. P.,unpublished).

Deamination of RNA nucleotides also occurs in the brain where RED1, adouble stranded RNA adenosine deaminase edits the channel determinantsite in glutamate receptor pre-mRNA. This site controls the Ca²⁺permeability of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid(AMPA) receptors. Another double stranded RNA adenosine deaminase,DRADA, edits a different site in the glutamate receptor pre-mRNA (Kim,U. et al. (1994) J. Biol. Chem. 269: 13480-13489). Glutamate receptorRNA editing is differentially regulated (Nutt, S. L. (1994) Neuroreport5: 1679-1683). Since glutamate receptors are essential for fastexcitatory neurotransmission, RNA editing may play a critical role innormal brain function and development. Furthermore, dysfunction of RNAediting may have neuropathological consequences and could be related toneurodegenerative diseases (Nutt, et al, supra). Evidence suggests thatRED1 and DRADA are members of a larger gene family of enzymes thatdeaminate nuclear transcripts and have distinct but overlappingsubstrate specificities (Melcher, T. et al. (1996) Nature 379: 460-464).

Neurofibromatosis type I is a common neurological disease in whichtissue derived from the embryonic neural crest is predisposed to thedevelopment of cancer. The NF1 stone encodes a tumor suppressor that isresponsible for neurofibromatosis type I. NF1 mRNA undergoes C to Uediting, creating an in-frame stop codon, in a manner similar to apoBmRNA editing. However, evidence suggests that the apoB complex does notfunction in NF1 mRNA editing (Skuse, G. R. et al. (1996) Nuc. Acids Res.24: 478-485). Cappione, A. J. et al. found that unusually high levels ofNF1 mRNA editing occur in tumor tissues associated withneurofibromatosis type I (1997; Am. J. Hum. Genet. 60: 305-312).

Discovery of proteins related to human RNA editing enzymes and thepolynucleotides encoding them satisfies a need in the art by providingnew compositions useful in diagnosis. prevention, and treatment ofcancer, autoimmune disorders, circulatory system disorders, viraldiseases, and neurological diseases.

SUMMARY OF THE INVENTION

The present invention features a novel human RNA editing enzymehereinafter designated PEE-2 and characterized as having similarity tothe mRNA editing enzymes phorbolin I. HEPR. and REPR.

Accordingly, the invention features a substantially purified REE-2having the amino acid sequence shown in SEQ ID NO:1.

One aspect of the invention features isolated and substantially purifiedpolynucleotides that encode REE-2. In a particular aspect, thepolynucleotide is the nucleotide sequence of SEQ ID NO:2.

The invention also relates to a polynucleotide sequence comprising thecomplement of SEQ ID NO:2 or variants thereof. In addition, theinvention features polynucleotide sequences which hybridize understringent conditions to SEQ ID NO:2.

The invention additionally features nucleic acid sequences encodingpolypeptides, oligonucleotides, peptide nucleic acids (PNA), fragments,portions or antisense molecules thereof, and expression vectors and hostcells comprising polynucleotides that encode REE-2. The presentinvention also features antibodies which bind specifically to REE-2, andpharmaceutical compositions comprising substantially purified REE-2. Theinvention also features agonists and antagonists of REE-2. The inventionalso features a method for producing REE-2, and methods for treatingcancer, circulatory system disorders, viral diseases, and neurologicaldiseases by administering REE-2 or an REE-2 fragment or derivativethereof.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the amino acid sequence (SEQ ID NO: 1) and nucleicacid sequence (SEQ ID NO:2) of REE-2. The alignment was produced usingMacDNASIS PRO™ software (Hitachi Software Engineering Co., Ltd., SanBruno, Calif.).

FIGS. 2A and 2B show the amino acid sequence alignments among REE-2 (SEQID NO:1), phorbolin I (GI 436941; SEQ ID NO:3), HEPR (GI 1177798; SEQ IDNO:4), and REPR (GI 585813; SEQ ID NO:5). The alignment was producedusing the multisequence alignment program of DNASTAR™ software (DNASTARInc. Madison Wis.).

FIG. 3 shows the hydrophobicity plot (MacDNASIS PRO software) for REE-2.SEQ ID NO: 1; the positive X axis reflects amino acid position, and thenegative Y axis, hydrophobicity.

FIG. 4 shows the hydrophobicity plot for HEPR, SEQ ID NO:4.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms "a", "an", and "the" include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to "ahost cell" includes a plurality of such host cells, reference to the"antibody" is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing the cell lines, vectors, and methodologies which are reportedin the publications which might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

Definitions

"Nucleic acid sequence", as used herein, refers to an oligonucleotide,nucleotide, or polynucleotide, and fragments or portions thereof, and toDNA or RNA of genomic or synthetic origin which may be single- ordouble-stranded, and represent the sense or antisiense strand.Similarly. "amino acid sequence", as used herein, refers to anoligopeptide, peptide, polypeptide, or protein sequence, and fragmentsor portions thereof, and to naturally occurring or synthetic molecules.

Where "amino acid sequence" is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, "amino acidsequence" and like terms, such as "polypeptide" or "protein" are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

"Peptide nucleic acid", as used herein, refers to a molecule whichcomprises an oligomer to which an amino acid residue, such as lysine,and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid (Nielsen, P. E. et al. (1993)Anticancer Drug Des. 8:53-63).

REE-2, as used herein, refers to the amino acid sequences ofsubstantially purified REE-2 obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

"Consensus", as used herein, refers to a nucleic acid sequence which hasbeen resequenced to resolve uncalled bases, or which has been extendedusing XL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5' and/or the 3'direction and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte clone using the GEL VIEW™Fragment Assembly system (GCG, Madison, Wis.), or which has been bothextended and assembled.

A "variant" of REE-2, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have"conservative" changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. More rarely, a variant may have "nonconservative" changes,e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

A "deletion", as used herein, refers to a change in either amino acid ornucleotide sequence in which one or more amino acid or nucleotideresidues, respectively, are absent.

An "insertion" or "addition", as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid or nucleotide residues, respectively, as compared to thenaturally occurring molecule.

A "substitution", as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

The term "biologically active", as used herein, refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, "immunologically active" refers to thecapability of the natural, recombinant, or synthetic REE-2, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The term "agonist", as used herein, refers to a molecule which, whenbound to REI -2, causes a change in REE-2 which modulates the activityof REE-2. Agonists may include proteins, nucleic acids, carbohydrates,or any other molecules which bind to REE-2.

The terms "antagonist" or "inhibitor", as used herein, refer to amolecule which, when bound to REE-2, blocks or modulates the biologicalor immunological activity of REE-2. Antagonists and inhibitors mayinclude proteins, nucleic acids, carbohydrates, or any other moleculeswhich bind to REE-2.

The term "modulate", as used herein, refers to a change or an alterationin the biological activity of REE-2. Modulation may be an increase or adecrease in protein activity, a change in binding characteristics, orany other change in the biological, functional or immunologicalproperties of REE-2.

The term "mimetic", as used herein, refers to a molecule, the structureof which is developed from knowledge of the structure of REE-2 orportions thereof and, as such, is able to effect some or all of theactions of RNA editing enzyme-like molecules.

The term "derivative", as used herein, refers to the chemicalmodification of a nucleic acid encoding REE-2 or the encoded REE-2.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics of thenatural molecule.

The term "substantially purified", as used herein, refers to nucleic oramino acid sequences that are removed from their natural environment,isolated or separated, and are at least 60% free, preferably 75% free,and most preferably 90% free from other components with which they arenaturally associated.

"Amplification" as used herein refers to the production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art(Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y.).

The term "hybridization", as used herein, refers to any process by whicha strand of nucleic acid binds with a complementary strand through basepairing.

The term "hybridization complex", as used herein, refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀ t or R₀ tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

The terms "complementary" or "complementarity", as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, for the sequence"A-G-T" binds to the complementary sequence "T-C-A". Complementaritybetween two single-stranded molecules may be "partial", in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acid strands.

The term "homology", as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term"substantially homologous." The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second noncomplementary target sequence.

As known in the art, numerous equivalent conditions may be employed tocomprise either low or high stringency conditions. Factors such as thelength and nature (DNA, RNA, base composition) of the sequence, natureof the target (DNA, RNA, base composition, presence in solution orimmobilization, etc.), and the concentration of the salts and othercomponents (e.g., the presence or absence of formamide, dextran sulfateand/or polyethylene glycol) are considered and the hybridizationsolution may be varied to generate conditions of either low or highstringency different from, but equivalent to, the above listedconditions.

The term "stringent conditions", as used herein, is the "stringency"which occurs within a range from about Tm-5° C. (5° C. below the meltingtemperature (Tm) of the probe) to about 20° C. to 25° C. below Tm. Aswill be understood by those of skill in the art, the stringency ofhybridization may be altered in order to identify or detect identical orrelated polynucleotide sequences.

The term "antisense", as used herein, refers to nucleotide sequenceswhich are complementary to a specific DNA or RNA sequence. The term"antisense strand" is used in reference to a nucleic acid strand that iscomplementary to the "sense" strand. Antisense molecules may be producedby any method, including synthesis by ligating the gene(s) of interestin a reverse orientation to a viral promoter which permits the synthesisof a complementary strand. Once introduced into a cell, this transcribedstrand combines with natural sequences produced by the cell to formduplexes. These duplexes then block either the further transcription ortranslation. In this manner, mutant phenotypes may be generated. Thedesignation "negative" is sometimes used in reference to the antisensestrand, and "positive" is sometimes used in reference to the sensestrand.

The term "portion", as used herein, with regard to a protein (as in "aportion of a given protein") refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid. Thus, a protein "comprising atleast a portion of the amino acid sequence of SEQ ID NO:1" encompassesthe full-length human REE-2 and fragments thereof.

"Transformation", as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but is not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such"transformed" cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

The term "antigenic determinant", as used herein, refers to that portionof a molecule that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The terms "specific binding" or "specifically binding", as used herein,in reference to the interaction of an antibody and a protein or peptide,mean that the interaction is dependent upon the presence of a particularstructure (i.e., the antigenic determinant or epitope) on the protein;in other words, the antibody is recognizing and binding to a specificprotein structure rather than to proteins in general. For example, if anantibody is specific for epitope "A", the presence of a proteincontaining epitope A (or free, unlabeled A) in a reaction containinglabeled "A" and the antibody will reduce the amount of labeled A boundto the antibody.

The term "sample", as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acid encoding REE-2 orfragments thereof may comprise a cell, chromosomes isolated from a cell(e.g., a spread of metaphase chromosomes), genomic DNA (in solution orbound to a solid support such as for Southern analysis), RNA (insolution or bound to a solid support such as for northern analysis),cDNA (in solution or bound to a solid support), an extract from cells ora tissue, and the like.

The term "correlates with expression of a polynucleotide", as usedherein, indicates that the detection of the presence of ribonucleic acidthat is similar to SEQ ID NO:2 by northern analysis is indicative of thepresence of mRNA encoding REE-2 in a sample and thereby correlates withexpression of the transcript from the polynucleotide encoding theprotein.

"Alterations" in the polynucleotide of SEQ ID NO:2, as used herein,comprise any alteration in the sequence of polynucleotides encodingREE-2 including deletions, insertions, and point mutations that may bedetected using hybridization assays. Included within this definition isthe detection of alterations to the genomic DNA sequence which encodesREE-2 (e.g., by alterations in the pattern of restriction fragmentlength polymorphisms capable of hybridizing to SEQ ID NO:2), theinability of a selected fragment of SEQ ID NO:2 to hybridize to a sampleof genomic DNA (e.g., using allele-specific oligonucleotide probes), andimproper or unexpected hybridization, such as hybridization to a locusother than the normal chromosomal locus for the polynucleotide sequenceencoding REE-2 (e.g., using fluorescent in situ hybridization [FISH] tometaphase chromosome spreads).

As used herein, the term "antibody" refers to intact molecules as wellas fragments thereof, such as Fa, F(ab')₂, and Fv, which are capable ofbinding the epitopic determinant. Antibodies that bind PEE-2polypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or peptide used to immunize an animal can be derived fromthe transition of RNA or synthesized chemically, and can be conjugatedto a carrier protein, if desired. Commonly used carriers that arechemically coupled to peptides include bovine serum albumin andthyroglobulin. The coupled peptide is then used to immunize the animal(e.g.. a mouse, a rat, or a rabbit).

The term "humanized antibody", as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

The Invention

The invention is based on the discovery of a novel human RNA editingenzyme, (REE-2), the polynucleotides encoding REE-2, and the use ofthese compositions for the diagnosis, prevention, or treatment ofcancer, autoimmune disorders, circulatory system disorders, viraldiseases, and neurological diseases.

Nucleic acids encoding the human REE-2 of the present invention werefirst identified in Incyte Clone 1646823 from the prostate tumor tissuecDNA library (PROSTUT09) through a computer-generated search for aminoacid sequence alignments. A consensus sequence, SEQ ID NO:2, was derivedfrom the following overlapping and/or extended nucleic acid sequences:Incyte Clones 1646823 (PROSTUT09), 1354426 (LUNGNOT09), and 1681742(STOMFET01).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A and 1B.REE-2 is 190 amino acids in length and has chemical and structuralhomology with phorbolin I (GI 436941; SEQ ID NO:3), HEPR (GI 1177798;SEQ ID NO:4), and REPR (GI 585813; SEQ ID NO:5; FIGS. 2A and 2B). Inparticular, REE-2 shares 28% identity with HEPR, 30% identity with REPR,and 43% identity with a partial sequence of phorbolin I. REE-2 containsconserved zinc-coordinating residues C₉₇ and C₁₀₀, and catalyticallyactive residues E₆₈ and P₉₆, necessary for both RNA editing and cytidinedeaminase activities. REE also contains conserved residue H₆₆, requiredfor RNA binding, RNA editing, and cytidine deaminase activities. Asillustrated by FIGS. 3 and 4, REE-2 and HEPR have rather similarhydrophobicity plots. Northern analysis revealed the expression of thissequence in various cDNA libraries, a high proportion of which arederived from tumors, neuronal tissues, immune system cells, or synovialtissue from arthritis patients.

The invention also encompasses REE-2 variants. A preferred REE-2 variantis one having at least 80%, and more preferably 90%, amino acid sequencesimilarity to the REE-2 amino acid sequence (SEQ ID NO:1). A mostpreferred REE-2 variant is one having at least 95% amino acid sequencesimilarity to SEQ ID NO:1.

The invention also encompasses polynucleotides which encode REE-2.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of REE-2 can be used to generate recombinant molecules whichexpress REE-2. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 asshown in FIGS. 1A and 1B.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding REE-2, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring REE-2, and all such variations are to beconsidered as being specifically disclosed.

Although nucleotide sequences which encode REE-2 and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring REE-2 under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding REE-2 or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding REE-2 and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences, or portionsthereof, which encode REE-2 and its derivatives, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art at the time of the filing ofthis application. Moreover, synthetic chemistry may be used to introducemutations into a sequence encoding REE-2 or any portion thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed nucleotide sequences, and inparticular, those shown in SEQ ID NO:2, under various conditions ofstringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe, as taughtin Wahl, G. M. and S. L. Berger (1987; Methods Enzymol. 152:399-407) andKimmel, A. R. (1987; Methods Enzymol. 152:507-511), and may be used at adefined stringency.

Altered nucleic acid sequences encoding REE-2 which are encompassed bythe invention include deletions, insertions, or substitutions ofdifferent nucleotides resulting in a polynucleotide that encodes thesame or a functionally equivalent REE-2. The encoded protein may alsocontain deletions, insertions, or substitutions of amino acid residueswhich produce a silent change and result in a functionally equivalentREE-2. Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of REE-2 is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid;positively charged amino acids may include lysine and arginine; andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine;phenylalanine and tyrosine.

Also included within the scope of the present invention are alleles ofthe genes encoding REE-2. As used herein, an "allele" or "allelicsequence" is an alternative form of the gene which may result from atleast one mutation in the nucleic acid sequence. Alleles may result inaltered mRNAs or polypeptides whose structure or function may or may notbe altered. Any given gene may have none, one, or many allelic forms.Commnon mutational changes which give rise to alleles are generallyascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

Methods for DNA sequencing which are well known and generally availablein the art may be used to practice any embodiments of the invention. Themethods may employ such enzymes as the Klenow fragment of DNA polymeraseI, Sequenase® (US Biochemical Corp. Cleveland, Ohio), Taq polymerase(Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, Ill.), orcombinations of recombinant polymerases and proofreading exonucleasessuch as the ELONGASE Amplification System marketed by Gibco BRL(Gaithersburg, Md.). Preferably, the process is automated with machinessuch as the Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), PeltierThermal Cycler (PTC200; MJ Research, Watertown, Mass.) and the ABI 377DNA sequencers (Perkin Elmer).

The nucleic acid sequences encoding REE-2 may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,"restriction-site" PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to linker sequence and a primer specific to the knownregion. The amplified sequences are then subjected to a second round ofPCR with the same linker primer and another specific primer internal tothe first one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed using OLIGO4.06 Primer Analysis software (National Biosciences Inc., Plymouth,Minn.), or another appropriate program, to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68°-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

Another method which may be used is capture PCR which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR.

Another method which may be used to retrieve unknown sequences is thatof Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTER FINDER™libraries to walk in genomic DNA (Clontech, Palo Alto, Calf.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5' regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into the 5' and 3'non-transcribed regulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devicecamera. Output/light intensity may be converted to electrical signalusing appropriate software (e.g. GENOTYPER™ and SEQUENCE NAVIGATOR™,Perkin Elmer) and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for the sequencing ofsmall pieces of DNA which might be present in limited amounts in aparticular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode REE-2, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of REE-2 in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and these sequences may be used to clone and expressREE-2.

As will be understood by those of skill in the art, it may beadvantageous to produce REE-2-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce a recombinant RNAtranscript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter REE-2encoding sequences for a variety of reasons, including but not limitedto, alterations which modify the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, or introduce mutations, and so forth.

In another embodiment of the invention, natural, moified, or recombinantnucleic acid sequences encoding REE-2 may be ligated to a heterologoussequence to encode a fusion protein. For example, to screen peptidelibraries for inhibitors of REE-2 activity, it may be useful to encode achimeric REE-2 protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the REE-2 encoding sequence and theheterologous protein sequence, so that REE-2 may be cleaved and purifiedaway from the heterologous moiety.

In another embodiment, sequences encoding REE-2 may be synthesized inwhole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of REE-2, or a portion thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge. J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 431 A Peptide Synthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton. T.(1983) Proteins, Structures and Molecular Principles, W H Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g.. the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of REE-2, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

In order to express a biologically active REE-2, the nucleotidesequences encoding REE-2 or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding REE-2 andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

A variety of expression vector/host systems may be utilized to containand express sequences encoding REE-2. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The "control elements" or "regulatory sequences" are thosenon-translated regions of the vector--enhancers, promoters, 5' and 3'untranslated regions--which interact with host cellular proteins tocarry out transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT® phagemid (Stratagene,LaJolla, Calif.) or PSPORT1™ plasmid (Gibco BRL) and the like may beused. The baculoviris polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding REE-2,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for REE-2. For example, when largequantities of REE-2 are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBLUESCRIPT® (Stratagene), in which the sequence encoding REE-2 may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding REE-2 may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV (Takamatsu. N. (1987) EMBO J. 6:307-311). Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680. Broglie, R.et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) ResultsProbl. Cell Differ. 17:85-105). These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. Such techniques are described in a number of generallyavailable reviews (see, for example, Hobbs, S. or Murry, L. E. in McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New York,N.Y.; pp. 191-196).

An insect system may also be used to express REE-2. For example, in onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The sequences encoding REE-2 may becloned into a non-essential region of the virus, such as the polyhedringene, and placed under control of the polyhedrin promoter. Successfulinsertion of REE-2 will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which REE-2 may be expressed (Engelhard, E. K. et al. (1994)Proc. Nat. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding REE-2 may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing REE-2 in infected host cells (Logan, J. and Shenk,T. (1984) Proc. Natl. Acad. Sci. 81 :3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding REE-2. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding REE-2, its initiation codon, and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a portion thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressREE-2 may be transformed using expression vectors which may containviral origins of replication and/or endogenous expression elements and aselectable marker gene on the same or on a separate vector. Followingthe introduction of the vector, cells may be allowed to grow for 1-2days in an enriched media before they are switched to selective media.The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using tissue culturetechniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy. I. et al. (1980) Cell 22:817-23) geneswhich can be employed in tk or aprt cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al. (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfaron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartmnan,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyarins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding REE-2 isinserted within a marker gene sequence, recombinant cells containingsequences encoding REE-2 can be identified by the absence of marker genefinction. Alternatively, a marker gene can be placed in tandem with asequence encoding REE-2 under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding REE-2 and express REE-2 may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

The presence of polynucleotide sequences encoding REE-2 can be detectedby DNA-DNA or DNA-RNA hybridization or amplification using probes orportions or fragments of polynucleotides encoding REE-2. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding REE-2 to detect transformantscontaining DNA or RNA encoding REE-2. As used herein "oligonucleotides"or "oligomers" refer to a nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides, preferably about 15 to30 nucleotides, and more preferably about 20-25 nucleotides, which canbe used as a probe or amplimer.

A variety of protocols for detecting and measuring the expression ofREE-2, using either polyclonal or monoclonal antibodies specific for theprotein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson REE-2 is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding REE-2 includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, the sequences encoding REE-2, orany portions thereof may be cloned into a vector for the production ofan mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.);Promega (Madison Wis.); and U.S. Biochemical Corp., Cleveland, Ohio).Suitable reporter molecules or labels, which may be used, includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with nucleotide sequences encoding REE-2 may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeREE-2 may be designed to contain signal sequences which direct secretionof REE-2 through a prokaryotic or eukaryotic cell membrane. Otherrecombinant constructions may be used to join sequences encoding REE-2to nucleotide sequence encoding a polypeptide domain which willfacilitate purification of soluble proteins. Such purificationfacilitating domains include, but are not limited to, metal chelatingpeptides such as histidine-tryptophan modules that allow purification onimmobilized metals, protein A domains that allow purification onimmobilized immunoglobulin, and the domain utilized in the FLAGSextension/affintity purification system (Immunex Corp., Seattle, Wash.).The inclusion of cleavable linker sequences such as those specific forFactor XA or enterokinase (Invitrogen, San Diego, Calif.) between thepurification domain and REE-2 may be used to facilitate purification.One such expression vector provides for expression of a fusion proteincontaining REE-2 and a nucleic acid encoding 6 histidine residuespreceding a thioredoxin or an enterokinase cleavage site. The histidineresidues facilitate purification on IMIAC (immobilized metal ionaffinity chromatography as described in Porath, J. et al. (1992, Prot.Exp. Purif. 3: 263-281)) while the enterokinase cleavage site provides ameans for purifying REE-2 from the fusion protein. A discussion ofvectors which contain fusion proteins is provided in Kroll, D. J. et al.(1993; DNA Cell Biol. 12:441-453).

In addition to recombinant production, fragments of REE-2 may beproduced by direct peptide synthesis using solid-phase techniques(Merrifield J. (1963) l. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 431 APeptide Synthesizer (Perkin Elmer). Various fragments of REE-2 may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule.

Therapeutics

Based on the chemical and structural homology among REE-2. phorbolin I,HEPR, and REPR; and the expression of REE-2 in tissue derived fromcancer patients, REE-2 appears to be associated with the development ofcancer, autoimmune disorders, circulators system disorders, viraldiseases, and neurological diseases.

Directed RNA editing may be used to alter transcripts of mutated genesthat are responsible for cancerous growth. Therefore, in one embodiment,REE-2 or a fragment or derivative thereof may be administered to asubject to treat or prevent cancer. Types of cancer include, but are notlimited to: adenocarcinoma; leukemia; melanoma; lymphoma; sarcoma; andcancers of the colon, liver, brain, small intestine, large intestine,breast, ovary, kidney, lung, and prostate.

In another embodiment, a vector capable of expressing REE-2, or afragment or a derivative thereof, may also be administered to a subjectto treat or prevent cancer including, but not limited, the types ofcancers listed above.

Directed RNA editing may be used to alter transcripts of viral genesthat are responsible for infectious diseases. Therefore, in oneembodiment, REE-2 or a fragment or derivative thereof may beadministered to a subject to treat or prevent viral diseases. Types ofviral diseases include, but are not limited to, those caused by HIV,hantavirus, poxyviruses, herpesviruses, papillomaviruses,polyomaviruses, adenoviruses, picornaviruses, and togaviruses.

In another embodiment, a vector capable of expressing REE-2, or afragment or a derivative thereof, may also be administered to a subjectto treat or prevent viral diseases including, but not limited, the typesof viral diseases listed above.

REE-2 is similar to phorbolin I, a protein whose expression rises in thecells of patients suffering from an autoimmune system disorder.Therefore, in one embodiment, an antagonist to REE-2 may be administeredto a subject to treat or prevent an autoimmune disorder. Such disordersinclude but are not limited to, rheumatoid arthritis, multiplesclerosis, scleroderma, psoriasis, Grave's disease, Sjogren's disease,Crohn's disease, diabetes, lupus, allergies, asthma, and myastheniagravis.

When adminitered to a patient, REE-2 may edit the transcripts of ApoB100 or other lipid transport proteins. This in turn may prevent theaccumulation of Apo B100 or other disease causing lipoproteins.Therefore, in one embodiment, REE-2 or a fragment or derivative thereofmay be administered to a subject to treat or prevent a circulatorysystem disorder. Such disorders include but are not limited to,atherosclerosis, hypercholesterolemia, hypocholesterolemia, and stroke.

In another embodiment, a vector capable of expressing REE-2, or afragment or a derivative thereof, may also be administered to a subjectto treat or prevent a circulatory system disorder including, but notlimited, the circulatory system disorders listed above.

A high level of RNA editing activity has been correlated with thepathogenesis of neurofibromatosis, a neurological disease. Therefore, inone embodiment, an antagonist to REE-2 may be administered to a subjectto treat or prevent a neurological disease. Such neurological diseasesinclude but are not limited to, neurofibromatosis, Alzheimer's disease,Parkinson's disease, neural tube defects, multiple sclerosis, dementia,and learning disabilities.

In other embodiments, any of the therapeutic proteins, antagonists,antibodies, agonists, antisense sequences or vectors described above maybe administered in combination with other appropriate therapeuticagents. Selection of the appropriate agents for use in combinationtherapy may be made by one of ordinary skill in the art, according toconventional pharmaceutical principles. The combination of therapeuticagents may act synergistically to effect the treatment or prevention ofthe various disorders described above. Using this approach, one may beable to achieve therapeutic efficacy with lower dosages of each agent,thus reducing the potential for adverse side effects.

Antagonists or inhibitors of REE-2 may be produced using methods whichare generally known in the art. In particular, purified REE-2 may beused to produce antibodies or to screen libraries of pharmaceuticalagents to identify those which specifically bind REE-2.

The antibodies may be generated using methods that are well known in theart. Such antibodies may include, but are not limited to, polyclonal,monoclonal, chimeric, single chain, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizinig antibodies, (i.e.,those which inhibit dimer formation) are especially preferred fortherapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith REE-2 or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the peptides, fragments, or oligopeptides used toinduce antibodies to REE-2 have an amino acid sequence consisting of atleast five amino acids, and more preferably at least 10 amino acids. Itis also preferable that they are identical to a portion of the aminoacid sequence of the natural protein, and they may contain the entireamino acid sequence of a small, naturally occurring molecule. Shortstretches of REE-2 amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

Monoclonal antibodies to REE-2 may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. etal. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc.Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol.62:109-120).

In addition, techniques developed for the production of "chimericantibodies", the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. etal. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceREE-2-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobin libraries (BurtonD. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites for REE-2 mayalso be generated. For example, such fragments include, but are notlimited to, the F(ab')2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab')2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between REE-2 and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering REE-2 epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides encodingREE-2, or any fragment thereof, or antisense molecules, may be used fortherapeutic purposes. In one aspect, antisense to the polynucleotideencoding REE-2 may be used in situations in which it would be desirableto block the transcription of the mRNA. In particular, cells may betransformed with sequences complementary to polynucleotides encodingREE-2. Thus, antisense molecules may be used to modulate REE-2 activity,or to achieve regulation of gene function. Such technology is now wellknown in the art, and sense or antisense oligomers or larger fragments,can be designed from various locations along the coding or controlregions of sequences encoding REE-2.

Expression vectors derived from retro viruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisensemolecules complementary to the polynucleotides of the gene encodingREE-2. These techniques are described both in Sambrook et al. (supra)and in Ausubel et al. (supra).

Genes encoding REE-2 can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide orfragment thereof which encodes PEE-2. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector and even longer if appropriate replicationelements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning antisense molecules, DNA, RNA, or PNA, to the control regionsof the gene encoding REE-2, i.e., the promoters, enhancers, and introns.Oligonucleotides derived from the transcription initiation site, e.g.,between positions -10 and +10 from the start site, are preferred.Similarly, inhibition can be achieved using "triple helix" base-pairingmethodology. Triple helix pairing is usefull because it causesinhibition of the ability of the double helix to open sufficiently forthe binding of polymerases, transcription factors, or regulatorymolecules. Recent therapeutic advances using triplex DNA have beendescribed in the literature (Gee, J. E. et al. (1994) In: Huber, B. E.and B. I. Carr, Molecular and Immunologic Approaches, Futura PublishingCo., Mt. Kisco, N.Y.). The antisense molecules may also be designed toblock translation of mRNA by preventing the transcript from binding toribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding REE-2.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Antisense molecules and ribozymes of the invention may be prepared byany method known in the art for the synthesis of nucleic acid molecules.These include techniques for chemically synthesizing oligonucleotidessuch as solid phase phosphoramidite chemical synthesis. Alternatively,RNA molecules may be generated by in vitro and in vivo transcription ofDNA sequences encoding REE-2. Such DNA sequences may be incorporatedinto a wide variety of vectors with suitable RNA polymerase promoterssuch as T7 or SP6. Alternatively, these cDNA constructs that synthesizeantisense RNA constitutively or inducibly can be introduced into celllines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the moleculeor the use of phosphorethioate or 2' O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection and by liposome injections may beachieved using methods which are well known in the art.

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of REE-2, antibodies toREE-2, mimetics, agonists, antagonists, or inhibitors of REE-2. Thecompositions may be administered alone or in combination with at leastone other agent, such as stabilizing compound, which may be administeredin any sterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions may be administered to a patient alone, or in combinationwith other agents, drugs or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of REE-2, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example REE-2 or fragments thereof, antibodies of REE-2,agonists, antagonists or inhibitors of REE-2, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind REE-2 may beused for the diagnosis of conditions or diseases characterized byexpression of REE-2, or in assays to monitor patients being treated withREE-2, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for REE-2 includemethods which utilize the antibody and a label to detect REE-2 in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

A variety of protocols including ELISA, RIA, and FACS for measuringREE-2 are known in the art and provide a basis for diagnosing altered orabnormal levels of REE-2 expression. Normal or standard values for REE-2expression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toREE-2 under conditions suitable for complex formation. The amount ofstandard complex formation may be quantified by various methods, butpreferably by photometric means. Quantities of REE-2 expressed insubject sample control and disease, samples from biopsied tissues arecompared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingREE-2 may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, antisense RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofREE-2 may be correlated with disease. The diagnostic assay may be usedto distinguish between absence, presence, and excess expression ofREE-2, and to monitor regulation of REE-2 levels during therapeuticintervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding REE-2 or closely related molecules, may be used to identifynucleic acid sequences which encode REE-2. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5' regulatory region, or a less specific region,e.g., especially in the 3' coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding REE-2, alleles, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe REE-2 encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring REE-2.

Means for producing specific hybridization probes for DNAs encodingREE-2 include the cloning of nucleic acid sequences encoding REE-2 orREE-2 derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

Polynucleotide sequences encoding REE-2 may be used for the diagnosis ofconditions or diseases which are associated with expression of REE-2.Examples of such conditions or diseases include, but are not limited to,cancer of the prostate, bladder, pancreas, colon, thyroid, and brain;rheumatoid arthritis; and inflamed adenoids. The polynucleotidesequences encoding REE-2 may be used in Southern or northern analysis,dot blot, or other membrane-based technologies; in PCR technologies; orin dip stick, pIN, ELISA or chip assays utilizing fluids or tissues frompatient biopsies to detect altered REE-2 expression. Such qualitative orquantitative methods are well known in the art.

In a particular aspect, the nucleotide sequences encoding REE-2 may beuseful in assays that detect activation or induction of various cancers,particularly those mentioned above. The nucleotide sequences encodingREE-2 may be labeled by standard methods, and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the biopsied or extracted sample issignificantly altered from that of a comparable control sample, thenucleotide sequences have hybridized with nucleotide sequences in thesample, and the presence of altered levels of nucleotide sequencesencoding REE-2 in the sample indicates the presence of the associateddisease. Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of disease associated withexpression of REE-2, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, which encodes REE-2, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withthose from an experiment where a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for disease. Deviation between standard and subjectvalues is used to establish the presence of disease.

Once disease is established and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in the normal patient. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding REE-2 may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced from arecombinant source. Oligomers will preferably consist of two nucleotidesequences, one with sense orientation (5'->3') and another withantisense (3'<-5'), employed under optimized conditions foridentification of a specific gene or condition. The same two oligomers,nested sets of oligomers, or even a degenerate pool of oligomers may beemployed under less stringent conditions for detection and/orquantitation of closely related DNA or RNA sequences.

Methods which may also be used to quantitate the expression of REE-2include radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and standard curves onto which the experimentalresults are interpolated (Melby, P. C. et al. (1993) J. Immunol.Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 229-236).The speed of quantitation of multiple samples may be accelerated byrunning the assay in an ELISA format where the oligomer of interest ispresented in various dilutions and a spectrophotometric or calorimetricresponse gives rapid quantitation.

In another embodiment of the invention, the nucleic acid sequences whichencode REE-2 may also be used to generate hybridization probes which areuseful for mapping the naturally occurring genomic sequence. Thesequences may be mapped to a particular chromosome or to a specificregion of the chromosome using well known techniques. Such techniquesinclude FISH, FACS, or artificial chromosome constructions, such asyeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

FISH (as described in Verma et al. (1988) Human Chromosomes: A Manual ofBasic Techniques, Pergamon Press, New York, N.Y.) may be correlated withother physical chromosome mapping techniques and genetic map data.Examples of genetic map data carts be found in the 1994 Genome Issue ofScience (265:1981 f). Correlation between the location of the geneencoding REE-2 on a physical chromosomal map and a specific disease, orpredisposition to a specific disease, may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier, or affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers may be used for extending genetic maps. Often the placement of agene on the chromosome of another mammalian species, such as mouse, mayreveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to 11q22-23 (Gatti, R. A. etal. (1988) Nature 336:577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc. among normal, carrier, or affected individuals.

In another embodiment of the invention, REE-2, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenREE-2 and the agent being tested, may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to REE-2 large numbers ofdifferent small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with REE-2, or fragments thereof, and washed. Bound REE-2 isthen detected by methods well known in the art. Purified REE-2 can alsobe coated directly onto plates for use in the aforementioned drugscreening techniques. Alternatively, non-neutralizing antibodies can beused to capture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding REE-2 specificallycompete with a test compound for binding REE-2. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with REE-2.

In additional embodiments, the nucleotide sequences which encode REE-2may be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES I PROSTUT09 cDNA Library Construction

The PROSTUT09 cDNA library was constructed from prostate tumor obtainedfrom a 66-year-old Caucasian male. Surgery included a radicalprostatectomy, a radical cystectomy, and a urinary diversion to theintestine. The pathology report indicated an invasive grade 3 (of 3)transitional cell carcinoma located within the prostatic urethra, andextended to involve periprostatic glands and diffusely invade theprostatic parencyma anteriorly and posteriorly. All final surgicalmargins including ureters (left and right, after multiple re-excisions)and prostatic urethra were negative for tumor. In addition to extensiveinvolvement by transitional cell carcinoma, the right prostate containeda microscopic focus of adenocarcinoma, Gleason grade 3+2, which wasconfined to the prostate and showed no capsular penetration. Multipleright and left pelvic lymph nodes were negative for tumor. The patientwas diagnosed with a malignant neoplasm of the prostate. The patienthistory included a previous transurethral prostatectomy and neoplasm ofuncertain behavior of the lung. The patient history included benignhypertension, cerebrovascular disease, arteriosclerotic coronary arterydisease, and tobacco use. The patient was taking insulin and DYAZIDE®(diuretic/antihypertensive; SmithKline Beecham Pharmaceuticals,Philadelphia, Pa.) at the time of surgery. The patient's family historyincluded benign hypertension, cerebrovascular disease, andarteriosclerotic coronary artery disease in the patient's sibling, amalignant neoplasm of the breast in the patient's mother, a malignantneoplasm of the upper lobe of the lung in the patient's sibling, and aprimary tuberculosis infection in the patient's father.

The frozen tissue was homogenized and lysed using a BrinkmannHomogenizer Polytron PT-3000 (Brinkmann Instruments, Westbury, N.Y.) inguanidinium isothiocyanate solution. The lysate was centrifuged over a5.7 M CsCl cushion using an Beckman SW28 rotor in a Beckman L8-70MUltracentrifuge (Beckman Instruments) for 18 hours at 25,000 rpm atambient temperature. The RNA was extracted with acid phenol pH 4.7,precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol,resuspended in RNAse-free water, and DNase treated at 37° C. Extractionand precipitation were repeated as before. The mRNA was then isolatedwith the Qiagen Oligotex kit (QIAGEN, Inc., Chatsworth, Calif.) and usedto construct the cDNA libraries.

The mRNAs were handled according to the recommended protocols in theSuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (Cat.#18248-013, Gibco/BRL. Gaithersburg, Md.). cDNAs were fractionated on aSepharose CL4B column (Cat. #275105-01, Pharmacia), and those cDNAsexceeding 400 bp were ligated into pINCY I. The plasmid pINCY I wassubsequently transformed into DH5a™ competent cells (Cat. #18258-012,Gibco/BRL).

II Isolation and Sequencing of cDNA Clones

Plasmid DNA was released from the cells and purified using the REAL Prep96 Plasmid Kit (Catalog #26173, QIAGEN, Inc.). This kit enabled thesimultaneous purification of 96 samples in a 96-well block usingmulti-channel reagent dispensers. The recommended protocol was employedexcept for the following changes: 1) the bacteria were cultured in 1 mlof sterile Terrific Broth (Catalog #22711, Gibco/BRL) with carbenicillinat 25 mg/L and glycerol at 0.4%; 2) after inoculation, the cultures wereincubated for 19 hours and at the end of incubation, the cells werelysed with 0.3 ml of lysis buffer; and 3) following isopropanolprecipitation, the plasmid DNA pellet was resuspended in 0.1 ml ofdistilled water. After the last step in the protocol, samples weretransferred to a 96-well block for storage at 4° C.

The cDNAs were sequenced by the method of Sanger et al. (1975, J. Mol.Biol. 94:441 f), using a Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.)in combination with Peltier Thermal Cyclers (PTC200 from MJ Research,Watertown, Mass.) and Applied Biosystems 377 DNA Sequencing Systems; andthe reading frame was determined.

III Homology Searching of cDNA Clones and Their Deduced Proteins

The nucleotide sequences of the Sequence Listing or amino acid sequencesdeduced from them were used as query sequences against databases such asGenBank, SwissProt, BLOCKS, and Pima II. These databases which containpreviously identified and annotated sequences were searched for regionsof homology (similarity) using BLAST, which stands for Basic LocalAlignment Search Tool (Altschul (1993) supra, Altschul (1990) supra).

BLAST produces alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST is especially useful in determining exact matches orin identifying homologs which may be of prokaryotic (bacterial) oreukaryotic (animal, fungal or plant) origin. Other algorithms such asthe one described in Smith R. F. and T. F. Smith (1992, ProteinEngineering 5:35-51). incorporated herein by reference, can be used whendealing with primary sequence patterns and secondary structure gappenalties. As disclosed in this application, the sequences have lengthsof at least 49 nucleotides, and no more than 12% uncalled bases (where Nis recorded rather than A, C, G, or T).

The BLAST approach, as detailed in Karlin and Altschul (supra) andincorporated herein by reference, searches for matches between a querysequence and a database sequence, to evaluate the statisticalsignificance of any matches found, and to report only those matcheswhich satisfy the user-selected threshold of significance. In thisapplication, threshold was set at 10⁻²⁵ for nucleotides and 10⁻¹⁴ forpeptides.

Incyte nucleotide sequences were searched against the GenBank databasesfor primate (pri), rodent (rod), and mammalian sequences (mam), anddeduced amino acid sequences from the same clones are searched againstGenBank functional protein databases, mamnmalian (mamp), vertebrate(vrtp) and eukaryote (eukp), for homology. The relevant database for aparticular match were reported as a GIxxx±p (where xxx is pri, rod, etcand if present, p=peptide). The product score is calculated as follows:the % nucleotide or amino acid identity [between the query and referencesequences] in BLAST is multiplied by the % maximum possible BLAST score[based on the lengths of query and reference sequences] and then dividedby 100. Where an Incyte Clone was homologous to several sequences, up tofive matches were provided with their relevant scores. In an analogy tothe hybridization procedures used in the laboratory, the electronicstringency for an exact match was set at 70, and the conservative lowerlimit for an exact match was set at approximately 40 (with 1-2% errordue to uncalled bases).

IV Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook et al., supra).

Analogous computer techniques using BLAST (Altschul, S. F. 1993 and1990. supra) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ™ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

The basis of the search is the product score which is defined as:##EQU1## The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding REE-2 occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V Extension of REE-2-Encoding Polynucleotides

Nucleic acid sequence of Incyte clone 1646823 or SEQ ID NO:2 is used todesign oligonucleotide primers for extending a partial nucleotidesequence to full length or for obtaining 5' or 3', intron or othercontrol sequences from genomic libraries. One primer is synthesized toinitiate extension in the antisense direction (XLR) and the other issynthesized to extend sequence in the sense direction (XLF). Primers areused to facilitate the extension of the known sequence "outward"generating amplicons containing new, unknown nucleotide sequence for theregion of interest. The initial primers are designed from the cDNA usingOLIGO 4.06 (National Biosciences), or another appropriate program, to be22-30 nucleotides in length, to have a GC content of 50% or more, and toanneal to the target sequence at temperatures about 68°-72° C. Anystretch of nucleotides which would result in hairpin structures andprimer-primer dimerizations is avoided.

The original, selected cDNA libraries, or a human genomic library areused to extend the sequence; the latter is most useful to obtain 5'upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

By following the instructions for the XL-PCR kit (Perkin Elmer) andthoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the Peltier Thermal Cycler (PTC200; M. J. Research,Watertown. Mass.) and the following parameters:

    ______________________________________                                        Step 1       94° C. for 1 min (initial denaturation)                                 Step 2 65° C. for 1 min                                    Step 3 68° C. for 6 min                                                Step 4 94° C. for 15 sec                                               Step 5 65° C. for 1 min                                                Step 6 68° C. for 7 min                                                Step 7 Repeat step 4-6 for 15 additional cycles                               Step 8 94° C. for 15 sec                                               Step 9 65° C. for 1 min                                                Step 10 68° C. for 7:15 min                                            Step 11 Repeat step 8-10 for 12 cycles                                        Step 12 72° C. for 8 min                                               Step 13 4° C. (and holding)                                          ______________________________________                                    

A 5-10, μl aliquot of the reaction mixture is analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products are selected and removedfrom the gel. Further purification involves using a commercial gelextraction method such as QIAQUICK™ (QIAGEN Inc., Chatsworth, Calif.).After recovery of the DNA, Klenow enzyme is used to trimsingle-stranded, nucleotide overhangs creating blunt ends whichfacilitate religation and cloning.

After ethanol precipitation, the products are redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C. the whole transformation mixtureis plated on Luria Bertani (LB)-agar (Sambrook et al., supra) containing2x Carb. The following day, several colonies are randomly picked fromeach plate and cultured in 150 μl of liquid LB/2x Carb medium placed inan individual well of an appropriate, commercially-available, sterile96-well microtiter plate. The following day, 5 μl of each overnightculture is transferred into a non-sterile 96-well plate and afterdilution 1:10 with water, 5 μl of each sample is transferred into a PCRarray.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3x)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction areadded to each well. Amplification is performed using the followingconditions:

    ______________________________________                                        Step 1     94° C. for 60 sec                                             Step 2 94° C. for 20 sec                                               Step 3 55° C. for 30 sec                                               Step 4 72° C. for 90 sec                                               Step 5 Repeat steps 2-4 for an additional 29 cycles                           Step 6 72° C. for 180 sec                                              Step 7 4° C. (and holding)                                           ______________________________________                                    

Aliquots of the PCR reactions are run on agarose gels together withmolecular weight markers. The sizes of the PCR products are compared tothe original partial cDNAs, and appropriate clones are selected, ligatedinto plasmid, and sequenced.

VI Labeling and Use of Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger cDNAfragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 μCi of [γ-³² P] adenosine triphosphate(Amersharn) and T4 polynucleotide kinase (DuPont NEN®, Boston, Mass.).The labeled oligonucleotides are substantially purified with SephadexG-25 superfine resin column (Pharmacia & Upjohn). A portion containing10⁷ counts per minute of each of the sense and antisenseoligonucleotides is used in a typical membrane based hybridizationanalysis of human genomic DNA digested with one of the followingendonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II; DUPONTNEN®).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham. N.H.) Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots in a Phosphoimagercassette (Molecular Dynamics, Sunnyvale, Calif.) for several hours,hybridization patterns are compared visually.

VII Antisense Molecules

Antisense molecules or the complement of the REE-2-encoding sequence, orany part thereof, is used to inhibit in vivo or in vitro expression ofnaturally occurring REE-2. Although use of antisense oligonucleotides,comprising about 20 base-pairs, is specifically described, essentiallythe same procedure is used with larger cDNA fragments. Anoligonucleotide based on the coding sequences of REE-2, as shown inFIGS. 1A and 1B, is used to inhibit expression of naturally occurringREE-2. The complementary oligonucleotide is designed from the mostunique 5' sequence as shown in FIGS. 1A and 1B and used either toinhibit transcription by preventing promoter binding to the upstreamnontranslated sequence or translation of an REE-2-encoding transcript bypreventing the ribosome from binding. Using an appropriate portion ofthe signal and 5' sequence of SEQ ID NO:2, an effective antisenseoligonucleotide includes any 15-20 nucleotides spanning the region whichtranslates into the signal or 5' coding sequence of the polypeptide asshown in FIGS. 1A and 1B.

VIII Expression of REE-2

Expression of REE-2 is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector, pSport, previously used for thegeneration of the cDNA library is used to express REE-2 in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

Induction of an isolated, transformed bacterial strain with IPTG usingstandard methods produces a fusion protein which consists of the firsteight residues of β-galactosidase, about 5 to 15 residues of linker, andthe fall length protein. The signal residues direct the secretion ofREE-2 into the bacterial growth media which can be used directly in thefollowing assay for activity.

IX Demonstration of REE-2 Activity

REE's deaminase activity can be measured by a method described byMacGinntie A. J. et al. (1995, J. Biol. Chem. 270: 14768-14775).Substantially purified REE is incubated with 3.3 uCi of [³ H]deoxycytidine and 250 uM cytidine in a total volume of 10 ul in a buffercontaining 45 mM TRIS, pH 7.5. After timed incubations the reaction isquenched by the addition of 2 ul of 10 ug/ul each deoxycytidine anddeoxyuridine. Any insoluble material is removed by centrifugation for 2minutes at fall speed in a microcentrifuge, and 4 ul of the reactionmixture is applied to a polyethyleneimine-cellulose thin layerchromatographic plate. The corresponding deoxycytidine and deoxyuridinebands are visualized by exposure to UV light and scraped intoscintillation fluid for quantification by liquid scintillationspectroscopy.

X Production of REE-2 Specific Antibodies

REE-2 that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 is analyzed using DNASTARsoftware (DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopolypeptide is synthesized and used to raiseantibodies by means known to those of skill in the art. Selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions, is described by Ausubel et al. (supra), and others.

Typically, the oligopeptides are 15 residues in length, synthesizedusing an Applied Biosystems Peptide Synthesizer Model 431A usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma,St. Louis, Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimideester (MBS; Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radioiodinated, goat anti-rabbitIgG.

XI Purification of Naturally Occurring REE-2 Using Specific Antibodies

Naturally occurring or recombinant REE-2 is substantially purified byimmunoaffinity chromatography using antibodies specific for REE-2. Animmunoaffinity column is constructed by covalently coupling REE-2antibody to an activated chromatographic resin, such as CnBr-activatedSepharose (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing REE-2 is passed over the imnmunoaffinity column, andthe column is washed under conditions that allow the preferentialabsorbance of REE-2 (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/REE-2 binding (eg, a buffer of pH 2-3 or a high concentrationof a chaotrope, such as urea or thiocyanate ion), and REE-2 iscollected.

XII Identification of Molecules Which Interact with REE-2

REE-2 or biologically active fragments thereof are labeled with ¹²⁵ IBolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled REE-2, washed and any wells withlabeled REE-2 complex are assayed. Data obtained using differentconcentrations of REE-2 are used to calculate values for the number,affinity, and association of REE-2 with the candidate molecules.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 5                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 190 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: PROSTUT09                                                        (B) CLONE: 1646823                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Met Asn Pro Gln Ile Arg Asn Pro Met Lys Al - #a Met Tyr Pro Gly        Thr                                                                              1               5  - #                10  - #                15              - - Phe Tyr Phe Gln Phe Lys Asn Leu Trp Glu Al - #a Asn Asp Arg Asn Glu                  20      - #            25      - #            30                   - - Thr Trp Leu Cys Phe Thr Val Glu Gly Ile Ly - #s Arg Arg Ser Val Val              35          - #        40          - #        45                       - - Ser Trp Lys Thr Gly Val Phe Arg Asn Gln Va - #l Asp Ser Glu Thr His          50              - #    55              - #    60                           - - Cys His Ala Glu Arg Cys Phe Leu Ser Trp Ph - #e Cys Asp Asp Ile Leu      65                  - #70                  - #75                  - #80        - - Ser Pro Asn Thr Lys Tyr Gln Val Thr Trp Ty - #r Thr Ser Trp Ser Pro                      85  - #                90  - #                95               - - Cys Pro Asp Cys Ala Gly Glu Val Ala Glu Ph - #e Leu Ala Arg His Ser                  100      - #           105      - #           110                  - - Asn Val Asn Leu Thr Ile Phe Thr Ala Arg Le - #u Tyr Tyr Phe Gln Tyr              115          - #       120          - #       125                      - - Pro Cys Tyr Gln Glu Gly Leu Arg Ser Leu Se - #r Gln Glu Gly Val Ala          130              - #   135              - #   140                          - - Val Glu Ile Met Asp Tyr Glu Asp Phe Lys Ty - #r Cys Trp Glu Asn Phe      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Val Tyr Asn Asp Asn Glu Pro Phe Lys Pro Tr - #p Lys Gly Leu Lys        Thr                                                                                             165  - #               170  - #               175             - - Asn Phe Arg Leu Leu Lys Arg Arg Leu Arg Gl - #u Ser Leu Gln                          180      - #           185      - #           190                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 610 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: PROSTUT09                                                        (B) CLONE: 1646833                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - ATGAATCCAC AGATCAGAAA CCCGATGAAG GCAATGTATC CAGGCACATT CT -             #ACTTCCAA     60                                                                 - - TTTAAAAACC TATGGGAAGC CAACGATCGG AACGAAACTT GGCTGTGCTT CA -            #CCGTGGAA    120                                                                 - - GGTATAAAGC GCCGCTCAGT TGTCTCCTGG AAGACGGGCG TCTTCCGAAA CC -            #AGGTGGAT    180                                                                 - - TCTGAGACCC ATTGTCATGC AGAAAGGTGC TTCCTCTCTT GGTTCTGCGA CG -            #ACATACTG    240                                                                 - - TCTCCTAACA CAAAGTACCA GGTCACCTGG TACACATCTT GGAGCCCTTG CC -            #CAGACTGT    300                                                                 - - GCAGGGGAGG TGGCCGAGTT CCTGGCCAGG CACAGCAACG TGAATCTCAC CA -            #TCTTCACC    360                                                                 - - GCCCGCCTCT ACTACTTCCA GTATCCATGT TACCAGGAGG GGCTCCGCAG CC -            #TGAGTCAG    420                                                                 - - GAAGGGGTCG CTGTGGAGAT CATGGACTAT GAAGATTTTA AATATTGTTG GG -            #AAAACTTT    480                                                                 - - GTGTACAATG ATAATGAGCC ATTCAAGCCT TGGAAGGGAT TAAAAACCAA CT -            #TTCGACTT    540                                                                 - - CTGAAAAGAA GGCTACGGGA GAGTCTCCAG TGAGGGGTCT CCCTGGGCCT CA -            #TGGTCTGT    600                                                                 - - CTCCTCTAAG                - #                  - #                      - #       610                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 116 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 436941                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Asn Ser Ala Arg Glu Ile Tyr Arg Val Thr Tr - #p Phe Ile Ser Trp Ser       1               5  - #                10  - #                15               - - Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Va - #l Arg Ala Phe Leu Gln                  20      - #            25      - #            30                   - - Glu Asn Thr His Val Arg Leu Pro Ile Phe Al - #a Ala Arg Ile Tyr Asp              35          - #        40          - #        45                       - - Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln Me - #t Leu Arg Asp Ala Gly          50              - #    55              - #    60                           - - Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Ph - #e Glu Tyr Cys Trp Asp      65                  - #70                  - #75                  - #80        - - Thr Phe Val Tyr Arg Gln Gly Cys Pro Phe Gl - #n Pro Trp Asp Gly Leu                      85  - #                90  - #                95               - - Glu Glu His Ser Gln Ala Leu Ser Gly Arg Le - #u Arg Ala Ile Leu Gln                  100      - #           105      - #           110                  - - Asn Gln Gly Asn                                                                  115                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 236 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 11777906                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Met Thr Ser Glu Lys Gly Pro Ser Thr Gly As - #p Pro Thr Leu Arg Arg       1               5  - #                10  - #                15               - - Arg Ile Glu Pro Trp Glu Phe Asp Val Phe Ty - #r Asp Pro Arg Glu Leu                  20      - #            25      - #            30                   - - Arg Lys Glu Ala Cys Leu Leu Tyr Glu Ile Ly - #s Trp Gly Met Ser Arg              35          - #        40          - #        45                       - - Lys Ile Trp Arg Ser Ser Gly Lys Asn Thr Th - #r Asn His Val Glu Val          50              - #    55              - #    60                           - - Asn Phe Ile Lys Lys Phe Thr Ser Glu Arg As - #p Phe His Pro Ser Ile      65                  - #70                  - #75                  - #80        - - Ser Cys Ser Ile Thr Trp Phe Leu Ser Trp Se - #r Pro Cys Trp Glu Cys                      85  - #                90  - #                95               - - Ser Gln Ala Ile Arg Glu Phe Leu Ser Arg Hi - #s Pro Gly Val Thr Leu                  100      - #           105      - #           110                  - - Val Ile Tyr Val Ala Arg Leu Phe Trp His Me - #t Asp Gln Gln Asn Arg              115          - #       120          - #       125                      - - Gln Gly Leu Arg Asp Leu Val Asn Ser Gly Va - #l Thr Ile Gln Ile Met          130              - #   135              - #   140                          - - Arg Ala Ser Glu Tyr Tyr His Cys Trp Arg As - #n Phe Val Asn Tyr Pro      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Pro Gly Asp Glu Ala His Trp Pro Gln Tyr Pr - #o Pro Leu Trp Met        Met                                                                                             165  - #               170  - #               175             - - Leu Tyr Ala Leu Glu Leu His Cys Ile Ile Le - #u Ser Leu Pro Pro Cys                  180      - #           185      - #           190                  - - Leu Lys Ile Ser Arg Arg Trp Gln Asn His Le - #u Thr Phe Phe Arg Leu              195          - #       200          - #       205                      - - His Leu Gln Asn Cys His Tyr Gln Thr Ile Pr - #o Pro His Ile Leu Leu          210              - #   215              - #   220                          - - Ala Thr Gly Leu Ile His Pro Ser Val Ala Tr - #p Arg                      225                 2 - #30                 2 - #35                            - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 229 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 585813                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Met Ser Ser Glu Thr Gly Pro Val Ala Val As - #p Pro Thr Leu Arg Arg       1               5  - #                10  - #                15               - - Arg Ile Glu Pro His Glu Phe Glu Val Phe Ph - #e Asp Pro Arg Glu Leu                  20      - #            25      - #            30                   - - Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile As - #n Trp Gly Gly Arg His              35          - #        40          - #        45                       - - Ser Ile Trp Arg His Thr Ser Gln Asn Thr As - #n Lys His Val Glu Val          50              - #    55              - #    60                           - - Asn Phe Ile Glu Lys Phe Thr Thr Glu Arg Ty - #r Phe Cys Pro Asn Thr      65                  - #70                  - #75                  - #80        - - Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Se - #r Pro Cys Gly Glu Cys                      85  - #                90  - #                95               - - Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg Ty - #r Pro His Val Thr Leu                  100      - #           105      - #           110                  - - Phe Ile Tyr Ile Ala Arg Leu Tyr His His Al - #a Asp Pro Arg Asn Arg              115          - #       120          - #       125                      - - Gln Gly Leu Arg Asp Leu Ile Ser Ser Gly Va - #l Thr Ile Gln Ile Met          130              - #   135              - #   140                          - - Thr Glu Gln Glu Ser Gly Tyr Cys Trp Arg As - #n Phe Val Asn Tyr Ser      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pr - #o His Leu Trp Val        Arg                                                                                             165  - #               170  - #               175             - - Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Le - #u Gly Leu Pro Pro Cys                  180      - #           185      - #           190                  - - Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Le - #u Thr Phe Phe Thr Ile              195          - #       200          - #       205                      - - Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pr - #o Pro His Ile Leu Trp          210              - #   215              - #   220                          - - Ala Thr Gly Leu Lys                                                      225                                                                          __________________________________________________________________________

What is claimed is:
 1. A method for detection of a polynucleotideencoding a polypeptide comprising SEO ID NO:1 in a biological sample,said method comprising the steps of:a) hybridizing an isolated andpurified polynucleotide which is complementary to a polynucleotideencoding a polypeptide comprising SEQ ID NO:1 to nucleic acid materialof a biological sample, thereby forming a hybridization complex; and b)detecting said hybridization complex, wherein the presence of saidcomplex correlates with the presence of a polynucleotide encoding apolypeptide comprising SEQ ID NO:1 in said biological sample.
 2. Themethod of claim 1, wherein the nucleic acid material of the biologicalsample is amplified by the polymerase chain reaction prior to thehybridization step.
 3. The method of claim 1, wherein the method iscarried out in a high-throughput format.
 4. A kit comprising an isolatedand purified polynucleotide comprising the polynucleotide sequence ofSEQ ID NO:2 or its complement, a detectable label, and means fordetecting said label.
 5. A method of detecting a target polynucleotidein a biological sample, said target polynucleotide comprising thesequence of SEQ ID NO;2, said method comprising:a) combining thebiological sample with a probe comprising at least 20 contiguousnucleotides, said probe comprising a sequence that is complementary tosaid target polynucleotide in the biological sample, under conditionssuitable for formation of a hybridization complex between said probe andsaid target polynucleotide; and b) detecting said hybridization complex,wherein the detection of said hybridization complex is correlated withthe presence of said target polynucleotide in the biological sample.